Probes containing DNA sequences which are complementary to specific chromosomal alphoid DNA are known to be useful as enumerators in in situ hybridization assays. The best known members of the prior art alphoid DNA sequences preparation methods use a common approach for isolating the alphoid DNA sequences. An enrichment based on a physical characteristic of repeated DNA is applied, DNA from the enriched pool is cloned and individual clones from this enriched pool are individually analyzed for utility in in situ hybridization assays. Searching such a pool has proven to be an inefficient and unreliable method for obtaining a sequence with high chromosomal specificity for a predetermined chromosome. The first such scheme to obtain cloned alphoid DNA used the buoyant density characteristics of alphoid DNA to produce an enriched pool of DNA sequences. (See Manuelidis, L., et al. in Chromosoma 66:23-32 (1978)). Other schemes for obtaining alphoid DNA clones have used the distribution of DNA restriction sites or the rapid renaturation of alphoid DNA relative to non-repeated species in the genome as the basis for producing enriched pools of alphoid DNA. (See Yang, T. P., et al., Proc. Natl. Acad. Sci. USA. 79:6593-6597 (1982), and Moyzis, R. K., et al., Chromosoma 95:375-386 (1987)). However, these methods are inherently relative inefficient and are not well suited for rapid commercial development of enumerator probes.
Also, prior art probes prepared from such sequences were indirect label probes and so required post-hybridization processing in order to achieve hybrid detection in contrast to direct label probes which require, for example, only one probe penetration step of a slide mounted specimen during in situ hybridization. Indirect label probes require the successful diffusion into the slide mounted specimen of the various protein reagents (antibodies, avidins, enzymes and the like) during an in situ hybridization multi-step procedure.
Prior art methods for labeling such prior art chromosome regionally specific complementary DNA sequences present difficulties in controlling the number of label moieties attached to individual sequences.
Improved DNA segments which are complementary to specific chromosomal DNA repeated segments existing in a particular chromosomal region, such as, for example, alphoid DNA in a specific chromosome, and improved methodology for making direct labeled probes therefrom, would be very useful. The present invention provides both such segments and such methodology.